Understanding the fundamental molecular mechanisms by which proteins fold remains one of the most challenging problems in structural biology. It is generally accepted that all of the information required for correct folding is contained within the amino acid sequence, but just how that "code" is translated into folding pathways and the unique three-dimensional structure required for biological activity is not yet known. There is an urgent need for direct experimental information on the structures and dynamics of the numerous conformational states populated in the folding landscape. There is also a need for more detailed understanding of the structure of kinetic folding intermediates and transition state ensembles, and of the fundamental molecular interactions that influence their rates of formation and determine their stability. The overall objective of the proposed research is to address these outstanding issues through kinetic and equilibrium studies of intermediates that populate the folding energy landscape of apomyoglobin. Apomyoglobin provides unique opportunities for detailed investigations of protein folding mechanisms since it exhibits relatively straightforward folding kinetics with well-defined folding intermediates and forms an equilibrium molten globule, similar in structure to the kinetic intermediate, under conditions that allow detailed NMR analysis. In addition, apomyoglobin forms a number of less structured states in acid solution that provide insights into the upper regions of the folding funnel. Multifunctional NMR methods will be used to investigate the structure and dynamics of the pH 4 molten globule, the pH 3 E state, and the pH 2 acid unfolded state of apomyoglobin. These studies will provide insights into the changes in structure and dynamics that accompany chain compaction, at a level of detail that cannot be obtained through kinetic experiments. Mutagenesis coupled with stopped flow kinetics and hydrogen exchange pulse labeling will be used to determine the molecular interactions that stabilize the kinetic molten globule intermediate and the transition state ensemble and influence their structures. This combined NMR and kinetic approach will allow mapping of a protein folding landscape at an unprecedented level of detail.